Cellular signaling networks are enormously complicated, in the whole consisting of highly regulated, branched and convergent cascades of interactions among thousands of components. Our goal is to dissect, thoroughly, a relatively small and tractable signaling network. However, multiple isoforms of individual components confuse the picture enormously. In some cases this may represent redundancy; in others it is clear that unique attributes of individual proteins endow the system with specific functionalities. We will approach these issues by examination of a few G protein-regulated pathways that we understand reasonably well, focusing on outputs high in the signaling pathway that can be quantified with available assays in a single cell type that is technologically tractable. Although this is still a very challenging problem, it cannot and must not be avoided. The feasibility of such approaches needs to be tested systematically and preferably in a single cell type where a critical mass of data can be gathered. The cell to be studied is HeLa. It is human and thus there is abundant information about its genome. At least a few different receptors regulate adenylyl cyclase activity bidirectionally in these cells, and different receptors regulate phospholipase Cb activity. Gene expression is manipulable with RNAi, the cells are easy to transfect, and they are amenable to modern imaging techniques. Specific aims include the following: (1) Define the parts list for regulation of adenylyl cyclase and phospholipase Cb activities in HeLa cells by G protein-coupled receptors (GPCRs). (2) Map the interactions between these components using a variety of techniques, with particular emphasis on perturbation of the system with RNAi and assessment of molecular interactions with fluorescence resonance energy transfer (FRET) techniques and by immunoprecipitation. (3) Probe mechanisms of restricted interactions between components of the system. (4) Quantify information flow through the system to the point of synthesis of the first stable second messenger (cyclic AMP and/or inositol 1,4,5 tris-phosphate [IP3]). An additional project relates to a newly discovered non-receptor guanine nucleotide exchange factor for certain G protein a subunits; this protein is known as Ric-8